1. Field of the Invention
This invention relates to a method of detecting a mismatching region, in which a mismatch binding has occurred, on a biochemical analysis micro array used for detection, analysis, or the like, of a specific sequence contained in a polynucleotide, such as a DNA.
2. Description of the Related Art
DNA micro arrays are expected to be applied to a wide range of fields of life science, such as monitoring of genetic expression, determination of base sequences of genes, analysis of gene polymorphism (SNP), analysis of gene amplification or deletion at cancered parts, classification of diseases, such as cancers, prediction of drug response characteristics, and searching of disease genes.
Principles of assay techniques utilizing DNA micro arrays are based upon detection of nucleic acids through hybridization. Specifically, various different probe DNA's are arrayed at a high density at a plurality of regions of a surface of a supporting material, such as glass, silicon, or a membrane filter, and secured to the regions of the surface of the supporting material. Thereafter, a target DNA (i.e., a DNA having been labeled with a labeling substance) is subjected to hybridization with the probe DNA's having been fixed to the regions of the surface of the supporting material. Signals obtained from the regions (spots) are then detected in the manner described below.
For example, in cases where the target DNA has been labeled with a radioactive labeling substance, a stimulable phosphor layer of a stimulable phosphor sheet is exposed to radiation radiated out from the radioactive labeling substance, which is contained selectively in the regions of the supporting material. Thereafter, the stimulable phosphor layer is exposed to stimulating rays, which cause the stimulable phosphor layer to emit light in proportion to the amount of energy stored on the stimulable phosphor layer during the exposure of the stimulable phosphor layer to the radiation. The light emitted by the stimulable phosphor layer is detected photoelectrically. In this manner, the target DNA having been specifically bound to at least one of the probe DNA'S, which have been fixed to the regions of the surface of the supporting material, is detected.
In cases where the target DNA has been labeled with a fluorescent labeling substance, excitation light is irradiated to the regions of the supporting material, and the fluorescent labeling substance, which is contained selectively in the regions of the supporting material, is excited by the excitation light to produce fluorescence. The thus produced fluorescence is detected photoelectrically.
In cases where the target DNA has been labeled with a chemical luminescent labeling substance capable of producing the chemical luminescence when being brought into contact with a chemical luminescence substrate, the chemical luminescent labeling substance, which is contained selectively in the regions of the supporting material, is brought into contact with the chemical luminescence substrate. Also, the chemical luminescence produced by the chemical luminescent labeling substance is detected photoelectrically. (The aforesaid assay techniques are described in, for example, U.S. Patent Laid-Open No. 20020016009.)
The DNA micro arrays may be classified into two groups in accordance with the kinds of the DNA's, which are arrayed, and processes for producing the DNA micro arrays. One of the two groups is an oligonucleotide array produced with a process, wherein a light blocking plate referred to as a mask is overlaid on a silicon base plate, the silicon base plate is exposed to light via the mask by the utilization of photo-lithography, which is an exposure technique for semiconductors, the operation for exposing the silicon base plate to light via the mask is iterated, and DNA molecules are thereby superposed one by one on the base plate. (The oligonucleotide array produced with the process described above will hereinbelow be referred to simply as the oligonucleotide array.) The other group is a cDNA micro array produced with a process, wherein cDNA's having been subjected to PCR amplification previously are spotted onto slide glass by use of a thin pin, an ink jet technique, or the like.
Conditions (such as a temperature and a salt concentration) optimum for the hybridization may vary for the different kinds of the probe DNA's having been fixed respectively to the regions. However, it is not always possible to perform the reaction under the conditions optimum for each of the probe DNA's, which have been fixed respectively to the regions having been located at a high density. Therefore, ordinarily, the reaction is performed under the identical conditions with respect to all of the regions. Accordingly, it may often occur that a target DNA, which is not perfectly complementary to a certain probe DNA on the array and has a sequence similar to the perfectly complementary sequence, undergoes incorrect hybridization with the aforesaid certain probe DNA on the array. The incorrect hybridization described above is referred to as the mismatch binding. The target DNA, which has undergone the mismatch binding, causes noise to occur at the time of signal detection and adversely affects a detection accuracy.
In particular, the cDNA micro array is produced by directly subjecting the cDNA's, which have been isolated from cells of organisms, to the PCR amplification and fixed to the slide glass. The cDNA micro array is not produced by previously designing the probe DNA's so as not to undergo a mismatch bonding as in the cases of the oligonucleotide array. Therefore, the cDNA micro array has a high possibility that the probe DNA's on the cDNA micro array will under go the mismatch binding. Accordingly, in the cases of the cDNA micro array, the mismatch binding is suppressed through preparation of the probes located on the cDNA micro array. For such purposes, for example, sequences specific to a gene to be detected are selected, and oligo DNA's having been synthesized in accordance with the selected sequences are used as the probes.
However, in both the cases of the oligonucleotide array and the synthetic oligo array, it is not always possible to design such that the mismatch binding does not occur. Particularly, in cases where analysis is to be made with respect to a long gene, such as a cDNA, it is almost impossible to design such that the mismatch binding does not occur.
Attempts have been made to solve the problems with regard to the mismatch binding described above by, for example, finely adjusting the temperature or the pH value at the time of hybridization of a target DNA with probe DNA's having been fixed to an array. For example, a biochemical reaction detecting chip for the hybridization of a polynucleotide with oligonucleotide probes, with which biochemical reaction detecting chip the biochemical reaction is capable of being caused to occur at a temperature optimum for the hybridization at each of probe fixing surfaces, has been proposed in, for example, U.S. Patent Laid-Open No. 20020164778. The proposed biochemical reaction detecting chip is based upon characteristics concerning a melting out temperature (i.e., a Tm value) of a complementary strand binding of oligonucleotide probes. Specifically, under conditions of temperatures lower than the Tm value, background noise due to the mismatch binding increases. Also, under conditions of temperatures higher than the Tm value, it becomes difficult for the polynucleotide to undergo the binding with the probes. Therefore, with the proposed biochemical reaction detecting chip, a temperature, at which the polynucleotide is capable of undergoing the hybridization with a probe such that the mismatch binding does not occur, is adjusted at a value optimum for each of the probes.
Also, a technique for selectively separating and recovering a desired polynucleotide is proposed in, for example, U.S. Pat. No. 6,093,370. With the proposed technique for selectively separating and recovering a desired polynucleotide, oligonucleotide probes are fixed respectively to regions of a surface of a base plate, and polynucleotides are subjected to the hybridization with the oligonucleotide probes. Thereafter, only a specific region of the base plate is heated selectively, and only the polynucleotide, which has been complementarily bound to the probe, is separated from the probe.
However, with the technique for utilizing the biochemical reaction detecting chip proposed in U.S. Patent Laid-Open No. 20020164778, it is necessary that a plurality of islands are formed on the biochemical reaction detecting chip, and that the temperature adjustment is monitored finely for each of the islands. Also, in cases where the technique for selectively separating and recovering a desired polynucleotide, which is proposed in U.S. Pat No. 6,093,370, is utilized, it is necessary that only the specific region of the base plate is heated selectively. Further, the Tm values of the probes, which are contained in the islands or the regions described above, must be set previously at approximately identical values. As described above, with each of the technique for utilizing the biochemical reaction detecting chip proposed in U.S. Patent Laid-Open No. 20020164778 and the technique for selectively separating and recovering a desired polynucleotide, which is proposed in U.S. Pat. No. 6,093,370, complicated operations must be performed, and it is necessary for a specific apparatus to be utilized. Furthermore, in order for the mismatch binding to be suppressed, fine adjustment for raising or lowering the salt concentration in a liquid subjected to reaction must be made, and considerable labor and time are thus required. In cases where the hybridization is performed through adjustment of the pH value, the same problems as those described above arise.
As described above, in order for the mismatch binding during the hybridization to be suppressed, complicated adjustments must be performed, and considerable labor and time are required. Also, new problems occur in that, depending upon the conditions for the suppression of the mismatch binding, perfect match binding, i.e. perfect complementary binding, is weakened.
If a region, at which a target DNA having under gone the mismatch binding is located, is capable of being specified, for example, the thus specified region will be capable of being eliminated at the time of data analysis, and the detection accuracy will thus be capable of being kept high.